taste modifying protein from miracle fruit - science 1968

8/7/2019 Taste Modifying Protein From Miracle Fruit - Science 1968

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the gulf. The earthquake epicentersshow a remarkable parallelism with thefracture zones that form the prominentbathymetric and structural lineations.

Profiler records, crossing the twosouthernmost offsets (Fig. 3, V and W),emphasize the faulted nature of thebathymetric lineations. To the east ofthe fault bordering the southernmostoffset, the Mexican mainland marginis made up of a thick section of sedi-ments within which there is a prominentunconformity, with probably Tertiarysediments forming much of the section.West of the fault, beneath the activelyspreading section, only very thin sedi-ments cover a basement reflector (Fig.3, V).

the gulf. The earthquake epicentersshow a remarkable parallelism with thefracture zones that form the prominentbathymetric and structural lineations.

Profiler records, crossing the twosouthernmost offsets (Fig. 3, V and W),emphasize the faulted nature of thebathymetric lineations. To the east ofthe fault bordering the southernmostoffset, the Mexican mainland marginis made up of a thick section of sedi-ments within which there is a prominentunconformity, with probably Tertiarysediments forming much of the section.West of the fault, beneath the activelyspreading section, only very thin sedi-ments cover a basement reflector (Fig.3, V).

Fig. 4. Bathymetric chart of the Gulf ofCalifornia. Positions of fracture zones(solid black lines) are based on reflectionprofiles. The crest of the East Pacific Rise,based on magnetic and seismic-reflectionprofiles at the mouth of the gulf, ishatched. Proposed positions of spreadingcenters are shown by double black lines;epicenters (14), by open (Gutenberg andRichter) and by solid circles (Sykes).Positions and directions of reflection pro-files of Fig. 3 are shown by solid arrows.20 SEPTEMBER 1968

Fig. 4. Bathymetric chart of the Gulf ofCalifornia. Positions of fracture zones(solid black lines) are based on reflectionprofiles. The crest of the East Pacific Rise,based on magnetic and seismic-reflectionprofiles at the mouth of the gulf, ishatched. Proposed positions of spreadingcenters are shown by double black lines;epicenters (14), by open (Gutenberg andRichter) and by solid circles (Sykes).Positions and directions of reflection pro-files of Fig. 3 are shown by solid arrows.20 SEPTEMBER 1968

Farther up the gulf, seismic-reflectionprofiles again give positive verificationof the faulted nature of the basin flanks.The section of profile X (Figs. 3 and4) shows faulting on the south side ofa spreading cell, and profile Y (Figs.3 and 4) shows a rifted trough definedby the bordering fracture zones. Thelatter profile also indicates that the nextfracture zone, well up the slope, pro-jects into the section as a distortedzone. Profile Z (Figs. 3 and 4) crossesthe mainland continental terrace to itsabrupt termination at a major easternboundary lineation; again the structureindicates that the next-northeasternfracture zone may be projected into thesection.

Two major faults that can be seencrossing the peninsula are the previous-ly documented Agua Blanca fault (15)and, to the south, a fault newly verified

by Fyfe (16); between them lies mostof the granite pluton of the peninsula.The Agua Blanca, in particular, isknown for its recent dextral movementsand, by our placement of spreadingaxes, would be a transform fault.

Additional verification of the trans-peninsular nature of the recently de-fined fault to the south is shown by thereflection profile T (Fig. 4) on thewest side of the peninsula. Fyfe foundthat the onshore fault turned more tothe west before heading out to sea.Projected on this course it coincidesneatly (Fig. 3) with the major struc-

tural discontinuity seen in the reflectionrecord.

On the basis of an axial growth rateof 3 cm/year and the distance of 120km from the present crest of the ridgeto the 1000-fathom (1830-m) isobathoff the tip of the peninsula, the pres-ent cycle of spreading began about4 million years ago. Because the spread-ing cell is believed to have migrated tothe west, translation of the peninsula,from near the west edge of the thickpelagic sediments to its present location,occurred at a doubled rate of about 6cm/year. Therefore, from about 4 to10 million years ago, during Late Mio-nene and Pliocene time, a proto-Gulfof California existed. The earlier periodof spreading of the sea floor, which isnow buried by the thick pelagic sedi-ments, also would have required about4 million years if the peninsula wasoriginally situated next to the mainland,

Farther up the gulf, seismic-reflectionprofiles again give positive verificationof the faulted nature of the basin flanks.The section of profile X (Figs. 3 and4) shows faulting on the south side ofa spreading cell, and profile Y (Figs.3 and 4) shows a rifted trough definedby the bordering fracture zones. Thelatter profile also indicates that the nextfracture zone, well up the slope, pro-jects into the section as a distortedzone. Profile Z (Figs. 3 and 4) crossesthe mainland continental terrace to itsabrupt termination at a major easternboundary lineation; again the structureindicates that the next-northeasternfracture zone may be projected into thesection.

Two major faults that can be seencrossing the peninsula are the previous-ly documented Agua Blanca fault (15)and, to the south, a fault newly verified

by Fyfe (16); between them lies mostof the granite pluton of the peninsula.The Agua Blanca, in particular, isknown for its recent dextral movementsand, by our placement of spreadingaxes, would be a transform fault.

Additional verification of the trans-peninsular nature of the recently de-fined fault to the south is shown by thereflection profile T (Fig. 4) on thewest side of the peninsula. Fyfe foundthat the onshore fault turned more tothe west before heading out to sea.Projected on this course it coincidesneatly (Fig. 3) with the major struc-

tural discontinuity seen in the reflectionrecord.

On the basis of an axial growth rateof 3 cm/year and the distance of 120km from the present crest of the ridgeto the 1000-fathom (1830-m) isobathoff the tip of the peninsula, the pres-ent cycle of spreading began about4 million years ago. Because the spread-ing cell is believed to have migrated tothe west, translation of the peninsula,from near the west edge of the thickpelagic sediments to its present location,occurred at a doubled rate of about 6cm/year. Therefore, from about 4 to10 million years ago, during Late Mio-nene and Pliocene time, a proto-Gulfof California existed. The earlier periodof spreading of the sea floor, which isnow buried by the thick pelagic sedi-ments, also would have required about4 million years if the peninsula wasoriginally situated next to the mainland,south of Puerto Vallarta, and movedto its Late Miocene or Early Plioceneposition at a rate comparable to that ofthe present cycle. The presence of rela-

south of Puerto Vallarta, and movedto its Late Miocene or Early Plioceneposition at a rate comparable to that ofthe present cycle. The presence of rela-

tively thick, probably Tertiary sedimentson the mainland continental margin ofMexico (Figs. 3 and 4: V, Y, and Z)complements the idea of existence of aLate Miocene-Pliocene gulf, and is ad-ditional evidence of the existence of aregional lapse in spreading of the seafloor during much of Pliocene time.

DAVID G. MOOREEDWIN C. BUFFINGTON

Marine Environment Division,Naval Undersea Warfare Center,San Diego, California 92132

References and Notes

1. Larson, Menard, and Smith's more recentindependent magnetic survey [Science 161,781 (1968)] of the East Pacific Rise, in the re-gion of its approaches to the gulf, resulted ina comprehensive chart of magnetic anomalieson and flanking the critical part of the riseas it approaches and enters the gulf. Theirstudies have reached several common con-clusions based on significantly different setsof data and are complementary.

2. H. W. Menard, Science 132, 1737 (1960);B. C. Heezen, Sci. Amer.

203,98

(1960).3. J. C. Crowell, Geol. Soc. Amer. Spec. Paper71 (1966).

4. G. A. Rusnak and R. L. Fisher, Amer. Assoc.Petrol. Geologists Mem. 3 (1964), p. 144; D. C.Krause, Bull. Geol. Soc. Amer. 76, 617 (1965).

5. W. Hamilton, Bull. Geol. Soc. Amer. 72,1307 (1961).6. F. J. Vine and D. H. Matthews, Nature 199,

947 (1963).7. J. T. Wilson, ibid. 207, 343 (1965).8. H. W. Menard, J. Geophys. Res. 71, 682

(1966).9. F. J. Vine, Science 154, 1405 (1966).

10. L. R. Sykes, in History of the Earth's Crust(NASA, in press).

11. W. R. Normark and J. R. Curray, Bull. Geol.Soc. Amer., in press.

12. A. Cox, R. R. Doell, G. B. Dalrymple,Science 144, 1537 (1964).13. D. A. Ross and G. G. Shor, J. Geophys. Res.

70, 5551 (1965).14. B. Gutenberg and C. F. Richter, Seismicity

of the Earth (Princeton Univ. Press, 1954);L. R. Sykes, J. Geophys. Res. 72, 2131 (1967).15. C. R. Allen, L. T. Silver, F. G. Stehli, Bull.Geol. Soc. Amer. 71, 467 (1960).

16. D. L. Fyfe, thesis, San Diego State College.17. Sponsored by NAVSHIPS under SR 104 03 01

(Task 0539).28 June 1968

Taste-Modifying Proteinfrom Miracle Fruit

Abstract. The active principle of mir-acle fruit (Synsepalum dulcificum) is abasic

glycoproteinwith a

probablemolecular weight of 44,000. Applica-tion of the protein to the tongue modi-fies the taste so that one tastes sour sub-stances as sweet.

A native shrub (Synsepalum dulcifi-cum) in tropical West Africa yields a

tively thick, probably Tertiary sedimentson the mainland continental margin ofMexico (Figs. 3 and 4: V, Y, and Z)complements the idea of existence of aLate Miocene-Pliocene gulf, and is ad-ditional evidence of the existence of aregional lapse in spreading of the seafloor during much of Pliocene time.

DAVID G. MOOREEDWIN C. BUFFINGTON

Marine Environment Division,Naval Undersea Warfare Center,San Diego, California 92132


1. Larson, Menard, and Smith's more recentindependent magnetic survey [Science 161,781 (1968)] of the East Pacific Rise, in the re-gion of its approaches to the gulf, resulted ina comprehensive chart of magnetic anomalieson and flanking the critical part of the riseas it approaches and enters the gulf. Theirstudies have reached several common con-clusions based on significantly different setsof data and are complementary.

2. H. W. Menard, Science 132, 1737 (1960);B. C. Heezen, Sci. Amer.

203,98

(1960).3. J. C. Crowell, Geol. Soc. Amer. Spec. Paper71 (1966).

4. G. A. Rusnak and R. L. Fisher, Amer. Assoc.Petrol. Geologists Mem. 3 (1964), p. 144; D. C.Krause, Bull. Geol. Soc. Amer. 76, 617 (1965).

5. W. Hamilton, Bull. Geol. Soc. Amer. 72,1307 (1961).6. F. J. Vine and D. H. Matthews, Nature 199,

947 (1963).7. J. T. Wilson, ibid. 207, 343 (1965).8. H. W. Menard, J. Geophys. Res. 71, 682

(1966).9. F. J. Vine, Science 154, 1405 (1966).

10. L. R. Sykes, in History of the Earth's Crust(NASA, in press).

11. W. R. Normark and J. R. Curray, Bull. Geol.Soc. Amer., in press.

12. A. Cox, R. R. Doell, G. B. Dalrymple,Science 144, 1537 (1964).13. D. A. Ross and G. G. Shor, J. Geophys. Res.

70, 5551 (1965).14. B. Gutenberg and C. F. Richter, Seismicity

of the Earth (Princeton Univ. Press, 1954);L. R. Sykes, J. Geophys. Res. 72, 2131 (1967).15. C. R. Allen, L. T. Silver, F. G. Stehli, Bull.Geol. Soc. Amer. 71, 467 (1960).

16. D. L. Fyfe, thesis, San Diego State College.17. Sponsored by NAVSHIPS under SR 104 03 01

(Task 0539).28 June 1968

Taste-Modifying Proteinfrom Miracle Fruit

Abstract. The active principle of mir-acle fruit (Synsepalum dulcificum) is abasic

glycoproteinwith a

probablemolecular weight of 44,000. Applica-tion of the protein to the tongue modi-fies the taste so that one tastes sour sub-stances as sweet.

A native shrub (Synsepalum dulcifi-cum) in tropical West Africa yields asmall, red berry that, once its pulp ischewed, causes sour substances to tastesweet. Local people often use it to maketheir stale and acidulated maize breadmore palatable and to give sweetness

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small, red berry that, once its pulp ischewed, causes sour substances to tastesweet. Local people often use it to maketheir stale and acidulated maize breadmore palatable and to give sweetness

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Table 1. Amino acid composition of taste-modifying protein. The data are given as resi-dues per 100 total residues. Three milli-grams of the lyophilized taste-modifyingprotein was hydrolyzed in 6N HC1 at 110?Cfor 22 hours. The amino acid analysis wasperformed on the Beckman model 120 aminoacid analyzer. Sugar components comprised6.7 percent of the protein as determined bythe tryptophan-sulfuric acid reaction (9).Paper chromatography was carried out by themethod of Masamune and Yosizawa (10) toidentify the sugar component. The sugars weredetected by the aniline hydrogen phthalate-spray method (11). Two red spots were iden-tified as L-arabinose and D-xylose, respectively,by comparison with known spots of sugars.

Amino acid Amino acidresidue per residue per

100 total residues 100 total residues

Lysine 7.9 Alanine 6.3Histidine 1.8 Half cystine 2.3(Ammonia) 17.4 Valine 8.0Arginine 4.7 Methionine 1.0Aspartic acid 11.3 Isoleucine 4.7Threonine 6.1 Leucine 6.5Serine 6.1 TryptophanGlutamic acid 9.2 Tyrosine 3.6Proline 6.0 Phenylalanine 5.0Glycine 9.8

to sour palm wine and beer. Daniell(1) first described the unusual proper-ties of the berry and called it miracu-lous berry. Others call it miracle fruit.

Inglett et al. (2) stated that "MiracleFruit has the unique property of caus-ing sour materials to taste sweet after

0.2 -o

0 i

2 .- _ , 0.

4 20 40 600 80 00 020l40v o0l m

Effluent volume (ml)

Fig. 1. Chromatography of taste-modifyingprotein on carboxymethyl-Sephadex C-25.A sample solution (100 ml) was appliedto a column (1.1 by 42 cm) of carboxy-methyl-Sephadex C-25 (100 to 270 mesh)buffered with 0.03M acetate buffer, pH 5.5.The column was eluted with a linear

gradient between 0.03M phosphate, pH6.0 (80 ml), and 0.2M phosphate, pH 8.8(80 ml); flow rate 20 ml/hour. The vol-ume of each fraction was 2.7 ml. Proteinwas detected by absorption at 280 nm.Sugar content was determined at 620 nmafter 0.25 ml of each fraction was coloredby reaction with the tryptophan-sulfuricacid. Activity of the protein was assayedwith 0.1 ml of each fraction after dilutionwith water to 5 ml, and expressed as theconcentration of sucrose whose sweetnessis equal to that of the 0.02M citric acidsolution. The yield of the protein from 300berries was 10 mg. Closed circles, protein;open circles, activity; and triangles, sugar.

1242

the mouth has been exposed to thefruit's mucilaginous material. Sourfoods, such as lemons, limes, grapefruit,rhubarb, and strawberries taste pleas-antly sweet; dilute organic and mineralacids also taste sweet." They attemptedto isolate the active principle of thefruit, but the active principle could notbe solubilized

by anytreatment in-

cluding extraction with water, salt solu-tion, or organic solvents or treatmentwith detergents or enzymes (2). Wenow report our studies on the isolationand characterization of the active prin-ciple from miracle fruit.

The berries were grown in a green-house at the Florida State University.Since the active principle is labile, theberries were stored in a deep freezer(-70?C) until needed. The sweeteningactivity was assayed on four subjects.Five milliliters of a solution containingthe active principle was kept in the

mouth for 2 minutes and was spit out.The mouth was rinsed with distilledwater, and 0.02M citric acid solutionwas tasted. For a quantitative measure-ment of the activity, the subject wasasked to choose one out of a series often sucrose solutions (0.1 to 1.OM)which best approximated the intensityof sweetness of the given citric acid.

The skin and seed of 300 berries wereremoved by hand, the pulp was ho-mogenized with distilled water, and thehomogenate was centrifuged (3). Theactive principle did not appear in the

supernatant.The

insoluble slurrywas

extracted with carbonate buffer (pH10.5). The extracts had a strong activ-ity, whereas the residue of the slurryhad no activity after the extraction pro-cedure was repeated three times. We

applied the combined extracts to a col-umn of diethylaminoethyl-Sephadex A-25 to remove colored materials (4).The effluent was colorless and had a

strong activity. The effluent, concen-trated with Aquacide (5), was used toobtain preliminary information on

properties of the active principle.Activity was destroyed when the

solution was boiled or exposed to ahigh concentration of organic solventsat room temperature. Activity wasgreatly decreased by exposure to pHabove 12.0 or below 2.5 at room tem-perature, whereas activity was stable at

pH 3.7 and 4?C for at least 1 month.When the solution was dialyzed againstdistilled water for 48 hours, no activitywas observed in the dialyzate. Additionof trypsin or pronase destroyed theactivity. These characteristics suggestthat the active principle is protein.

The molecular weight of the active

principle was estimated with a Sepha-dex G-75 column (6). The elution pat-tern of the active principle was typicalof that of a single homogeneous poly-mer. Elution volumes of proteins withknown molecular weights were deter-mined on the same column, and theobserved

elutionvolumes

were plottedagainst the logarithm of the molecularweights according to the method ofAndrews (7). The molecular weight ofthe active principle was estimated as44,000.

Further purification of the activeprinciple was carried out on a cation-exchange column. The concentratedeffluent from the diethylaminoethyl-Sephadex column was dialyzed against0.03M acetate buffer (pH 5.5) and ap-plied to a column of carboxymethyl-Sephadex C-25. The active principlewas absorbed on the column and eluted

with a gradient of phosphate buffer.The active fraction from the columnhad characteristics typical of protein;its reaction to biuret reagent was posi-tive, and its absorption maximum wasat 278 nm. The elution pattern of the

Fig. 2. Polyacrylamide disc electrophoresisof taste-modifying protein. Left, withouturea; right, in 8M urea. A concentratedactive fraction from carboxymethyl col-umn was applied to gels. Electrophoresiswas conducted at 4?C and 3 ma for 2hours at pH 4.5. The gels were stainedwith 1 percent amido black in 7.5 percentacetic acid solution.

SCIENCE, VOL. 161

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active protein, measured by ultravioletabsorption, correlates well with theactivity curve (Fig. 1). This purifiedprotein seems to be an aggregate of theprotein monomer, because applicationof the protein to the Sephadex G-75column indicated that the molecularweight of the purified protein, measured

by activity and ultraviolet absorption,was more than 80,000 (8). Aggregationproceeded during the dialyzing processagainst acetate buffer of low ionicstrength, although this process did notchange the activity of the protein.

Purity of the isolated protein waschecked with disc electrophoresis onpolyacrylamide gel. The protein mi-grated to the cathode at pH 4.5 (Fig.2) but did not migrate at pH 8.3. Thiselectrophoretic behavior was in agree-ment with that expected from the resultobtained with the cation exchange col-umn, and indicated that the protein is

basic. A single sharp band accompanieda light diffused band. These bandschanged into a single sharp band (Fig.2) when electrophoresis was carried outin 8M urea. This fact indicated that theisolated protein does not contain anyprotein other than the active com-ponent.

An amino-acid analysis of the proteinwas carried out after the lyophilizedprotein was hydrolyzed in 6N HC1(Table 1). Sugar components comprise6.7 percent of the protein. The sugarcontent of each fraction from a car-

boxymethyl-Sephadex column was de-termined by the tryptophan-sulfuric acidreaction (Fig. 1, triangles) (9). The pos-sibility that the sugars are an impuritywas eliminated by the fact that the curveof sugar content correlated well with thechromatographic pattern of the activeprotein (Fig. 1) and that the sugarcontent was not changed by dialysis orrechromatography of the fractions on acarboxymethyl-Sephadex column. Paperchromatography of the protein hy-drolyzate gave two sugar spots, whichwere identified as L-arabinose and D-xylose. These results indicated that the

active principle of miracle fruit is aglycoprotein.

The sweetening of the subsequenttaste of acids was observed at a 5 X10-8M concentration of the proteinsolution and reached a maximum at4 X 10-7M. The sweetening effect athigh concentration of the protein solu-

active protein, measured by ultravioletabsorption, correlates well with theactivity curve (Fig. 1). This purifiedprotein seems to be an aggregate of theprotein monomer, because applicationof the protein to the Sephadex G-75column indicated that the molecularweight of the purified protein, measured

by activity and ultraviolet absorption,was more than 80,000 (8). Aggregationproceeded during the dialyzing processagainst acetate buffer of low ionicstrength, although this process did notchange the activity of the protein.

Purity of the isolated protein waschecked with disc electrophoresis onpolyacrylamide gel. The protein mi-grated to the cathode at pH 4.5 (Fig.2) but did not migrate at pH 8.3. Thiselectrophoretic behavior was in agree-ment with that expected from the resultobtained with the cation exchange col-umn, and indicated that the protein is

basic. A single sharp band accompanieda light diffused band. These bandschanged into a single sharp band (Fig.2) when electrophoresis was carried outin 8M urea. This fact indicated that theisolated protein does not contain anyprotein other than the active com-ponent.

An amino-acid analysis of the proteinwas carried out after the lyophilizedprotein was hydrolyzed in 6N HC1(Table 1). Sugar components comprise6.7 percent of the protein. The sugarcontent of each fraction from a car-

boxymethyl-Sephadex column was de-termined by the tryptophan-sulfuric acidreaction (Fig. 1, triangles) (9). The pos-sibility that the sugars are an impuritywas eliminated by the fact that the curveof sugar content correlated well with thechromatographic pattern of the activeprotein (Fig. 1) and that the sugarcontent was not changed by dialysis orrechromatography of the fractions on acarboxymethyl-Sephadex column. Paperchromatography of the protein hy-drolyzate gave two sugar spots, whichwere identified as L-arabinose and D-xylose. These results indicated that the

active principle of miracle fruit is aglycoprotein.

The sweetening of the subsequenttaste of acids was observed at a 5 X10-8M concentration of the proteinsolution and reached a maximum at4 X 10-7M. The sweetening effect athigh concentration of the protein solu-tion slowly declines over a period up to2 hours. The purified protein itself hasno inherent taste. A mixture of theprotein with sour substances initiallytastes sour, and slowly changes to sweet

20 SEPTEMBER 1968

tion slowly declines over a period up to2 hours. The purified protein itself hasno inherent taste. A mixture of theprotein with sour substances initiallytastes sour, and slowly changes to sweet

20 SEPTEMBER 1968

if the mixture is held in the mouth forabout 1 minute. This fact ruled outthe possibility that a complex of theprotein with the sour substance itselfhas a sweet taste. It is believed that theprotein binds to receptors of the tastebuds and modifies their function. Wecall the protein "taste-modifying pro-

tein." KENZO KURIHARALLOYD M. BEIDLER

Department of Biological Science,Florida State University,Tallahassee


1. W. F. Daniell, Pharm. J. 11, 445 (1852).2. G. E. Inglett, B. Dowling, J. J. Albrecht, F.

A. Hoglan, J. Agr. Food Chem. 13, 284(1965).

3. Pulp (100 g) from 300 berries was homoge-nized with 200 ml of distilled water in aWaring blender for 1 minute. The homogenatewas centrifuged at 10,000 rev/min for 10minutes, and the sediment was washed withwater once more. The washed sediment washomogenized with 100 ml of 0.1M NaHCO3-Na2COa buffer, pH 10.5, for 1 minute. Afterthe homogenate was stirred for 5 minutes, itwas centrifuged at 10,000 rev/min. The ex-

if the mixture is held in the mouth forabout 1 minute. This fact ruled outthe possibility that a complex of theprotein with the sour substance itselfhas a sweet taste. It is believed that theprotein binds to receptors of the tastebuds and modifies their function. Wecall the protein "taste-modifying pro-

tein." KENZO KURIHARALLOYD M. BEIDLER

Department of Biological Science,Florida State University,Tallahassee


1. W. F. Daniell, Pharm. J. 11, 445 (1852).2. G. E. Inglett, B. Dowling, J. J. Albrecht, F.

A. Hoglan, J. Agr. Food Chem. 13, 284(1965).

3. Pulp (100 g) from 300 berries was homoge-nized with 200 ml of distilled water in aWaring blender for 1 minute. The homogenatewas centrifuged at 10,000 rev/min for 10minutes, and the sediment was washed withwater once more. The washed sediment washomogenized with 100 ml of 0.1M NaHCO3-Na2COa buffer, pH 10.5, for 1 minute. Afterthe homogenate was stirred for 5 minutes, itwas centrifuged at 10,000 rev/min. The ex-

ance of foreign protein.

The vertebrate immune response ischaracterized by (i) increased respon-siveness to a foreign substance as aresult of prior exposure to it, and (ii)specificity, permitting this response toproceed only against the immunizingsubstance and closely related substances.No invertebrate has yet been foundwhich possesses both of these two char-acteristics (1). Conceivably, duringevolutionary history, one of these char-acteristics might have developed beforethe other, and if so, one might expectto find animal species which have intheir response to foreign substances

only one of these characteristics. Re-cent evidence suggests that some in-

ance of foreign protein.

The vertebrate immune response ischaracterized by (i) increased respon-siveness to a foreign substance as aresult of prior exposure to it, and (ii)specificity, permitting this response toproceed only against the immunizingsubstance and closely related substances.No invertebrate has yet been foundwhich possesses both of these two char-acteristics (1). Conceivably, duringevolutionary history, one of these char-acteristics might have developed beforethe other, and if so, one might expectto find animal species which have intheir response to foreign substances

only one of these characteristics. Re-cent evidence suggests that some in-

traction was repeated three times with thecarbonate buffer. All of the preparative experi-ments in this paper were carried at 0 to 4?C.

4. Combined extracts with carbonate buffer wereapplied to a column (3.6 by 45 cm) ofDEAE-Sephadex A-25 (50 to 140 mesh)buffered with a 0.025M phosphate buffer, pHI6.0. The column was eluted with 0.1M carbo-nate buffer, pH 10.5, and 600 ml of activefraction was collected.

5. The pH of the effluent from DEAE-Sephadexcolumn was adjusted to 3.7, and the effluentwas placed in dialyzing tube. The powder of

Aquacide II (Calbiochem) was placed outsidethe tube and kept overnight. The volume (600ml) of the solution in the tube was concen-trated to 100 ml.

6. One milliliter of 0.5 percent protein solution(hemoglobin, chymotrypsin, ribonuclease, andcytochrome c) and a solution of taste-modi-fying protein was applied to a column (1.5by 61.5 cm) of Sephadex G-75 (0.1M phos-phate buffer, pH 7.0; flow rate 20 ml/hour).Activity was measured with 1 ml of eachfraction after dilution with water to 5 ml.

7. P. Andrews, Biochem. J. 91, 222 (1964).8. An ultracentrifugal pattern of the purified

protein showed a single peak, but the patterndiffused quickly.

9. J. Badin, C. Jackson, M. Shubert, Proc. Soc.Exp. Biol. Med. 84, 288 (1953).

10. H. Masamune and Z. Yosizawa, Tohoku J.Exp. Med. 59, 1 (1953).

11. S. M. Partridge, Nature 154, 443 (1949).12.

Supported byNSF

grantGB-4068X and AEC

grant AT-(40-1)-2690.

24 May 1968; revised 9 August 1968 U

traction was repeated three times with thecarbonate buffer. All of the preparative experi-ments in this paper were carried at 0 to 4?C.

4. Combined extracts with carbonate buffer wereapplied to a column (3.6 by 45 cm) ofDEAE-Sephadex A-25 (50 to 140 mesh)buffered with a 0.025M phosphate buffer, pHI6.0. The column was eluted with 0.1M carbo-nate buffer, pH 10.5, and 600 ml of activefraction was collected.

5. The pH of the effluent from DEAE-Sephadexcolumn was adjusted to 3.7, and the effluentwas placed in dialyzing tube. The powder of

Aquacide II (Calbiochem) was placed outsidethe tube and kept overnight. The volume (600ml) of the solution in the tube was concen-trated to 100 ml.

6. One milliliter of 0.5 percent protein solution(hemoglobin, chymotrypsin, ribonuclease, andcytochrome c) and a solution of taste-modi-fying protein was applied to a column (1.5by 61.5 cm) of Sephadex G-75 (0.1M phos-phate buffer, pH 7.0; flow rate 20 ml/hour).Activity was measured with 1 ml of eachfraction after dilution with water to 5 ml.

7. P. Andrews, Biochem. J. 91, 222 (1964).8. An ultracentrifugal pattern of the purified

protein showed a single peak, but the patterndiffused quickly.

9. J. Badin, C. Jackson, M. Shubert, Proc. Soc.Exp. Biol. Med. 84, 288 (1953).

10. H. Masamune and Z. Yosizawa, Tohoku J.Exp. Med. 59, 1 (1953).

11. S. M. Partridge, Nature 154, 443 (1949).12.

Supported byNSF

grantGB-4068X and AEC

grant AT-(40-1)-2690.

24 May 1968; revised 9 August 1968 U

vertebrates possess a degree of receptorspecificity toward antigens (2). We re-port here that the sea urchin (phylumEchinodermata), while not developinga state of increased responsiveness toforeign protein, nonetheless withoutprior exposure can respond specifically,discriminating between two structurallyrelated proteins. The finding of suchdiscriminative capacity suggests an evo-lutionary connection between this seaurchin response and the vertebrate im-mune response, a possibility whichseems more plausible because of theknown phylogenetic affinities of echino-

derms and chordates (3).Sea urchins Strongylocentrotus pur-

vertebrates possess a degree of receptorspecificity toward antigens (2). We re-port here that the sea urchin (phylumEchinodermata), while not developinga state of increased responsiveness toforeign protein, nonetheless withoutprior exposure can respond specifically,discriminating between two structurallyrelated proteins. The finding of suchdiscriminative capacity suggests an evo-lutionary connection between this seaurchin response and the vertebrate im-mune response, a possibility whichseems more plausible because of theknown phylogenetic affinities of echino-

derms and chordates (3).Sea urchins Strongylocentrotus pur-

Table 1. Clearance of radioactive molecules from the coelomic fluid of S. purpuratus.

No. of Protein present (ug/ml, ? 1 S.D.) Protein remainingMolecule animals at various times after injection after 22 hours

group 1 hour 13 hours 22 hours (mean percent)

BSA-C14 11 2.30 ? .24 0.70 ? .29 0.28 ? .15 12.2HSA-C14 6 2.97 + .44 0.96 + .19 0.31 ? .10 10.4S. purpuratus 4 2.55 ? .20 1.44 ?.26 1.36 ? .19 53.2

molecules** See text for preparation procedure.

1243

Table 1. Clearance of radioactive molecules from the coelomic fluid of S. purpuratus.

No. of Protein present (ug/ml, ? 1 S.D.) Protein remainingMolecule animals at various times after injection after 22 hours

group 1 hour 13 hours 22 hours (mean percent)

BSA-C14 11 2.30 ? .24 0.70 ? .29 0.28 ? .15 12.2HSA-C14 6 2.97 + .44 0.96 + .19 0.31 ? .10 10.4S. purpuratus 4 2.55 ? .20 1.44 ?.26 1.36 ? .19 53.2

molecules** See text for preparation procedure.

1243

Sea Urchin Response to Foreign Substances

Abstract. An assessment of the response of Strongylocentrotus purpuratus tosubstances injected into the coelom shows that (i) foreign molecules are clearedfrom the coelomic fluid more rapidly than native molecules, (ii) coelomocytes canrespond selectively to albumins of human and bovine origin, and (iii) immuniza-tion attempts fail to elicit accelerated coelomocyte uptake or accelerated clear-

Sea Urchin Response to Foreign Substances

Abstract. An assessment of the response of Strongylocentrotus purpuratus tosubstances injected into the coelom shows that (i) foreign molecules are clearedfrom the coelomic fluid more rapidly than native molecules, (ii) coelomocytes canrespond selectively to albumins of human and bovine origin, and (iii) immuniza-tion attempts fail to elicit accelerated coelomocyte uptake or accelerated clear-

taste modifying protein from miracle fruit - science 1968

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